Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease worldwide, affecting billions of the global population. It can gradually progress to more severe diseases, including steatohepatitis, cirrhosis and hepatocellular carcinoma. Studies have highlighted the importance of the gut microbiome in the pathogenesis and progression of MASLD. On the other hand, increasing evidence has revealed the clinical potential of targeting the gut microbiome to treat MASLD. In this Review, we summarize gut microbial alterations in MASLD, metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. The mechanisms by which a dysregulated gut-liver axis contributes to disease progression are also described, including intestinal barrier dysfunction, chronic inflammation, and altered metabolic pathways (for example, bile acids) and microbial-derived metabolites (for example, short-chain fatty acids, tryptophan derivatives and endogenous ethanol). In addition, we discuss the clinical implications of utilizing the gut microbiome as a diagnostic biomarker and the therapeutic approaches to treat MASLD and related diseases such as faecal microbiota transplantation, probiotics and engineered bacteria, prebiotics and postbiotics, microbial-derived metabolites, antimicrobials and bacteriophages. Finally, we discuss current challenges in basic and translational research on the microbiome in MASLD and propose future directions to drive progress in this field.